Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri

The anaerobic soil bacteriumEubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i ) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii ) two chiral intermediates were discovered, and (iii ) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded byndhF (FAD subunit),ndhS (2 x [2Fe-2S] subunit), and by thendhL /ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S]2+/1+ and two [4Fe-4S]2+/1+ clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S )-2-formylglutarate. NADH-dependent reduction of the latter to (S )-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B12 -dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R ,3S )-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of theE. barkeri nicotinate catabolism were identified in nine Proteobacteria.